Matsushita K, Yakushi T, Takaki Y, Toyama H, Adachi O
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
J Bacteriol. 1995 Nov;177(22):6552-9. doi: 10.1128/jb.177.22.6552-6559.1995.
Alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound quinohemoprotein-cytochrome c complex involved in vinegar production. In Gluconobacter suboxydans grown under acidic growth conditions, it was found that ADH content in the membranes was largely increased but the activity was not much changed, suggesting that such a condition produces an inactive form of ADH (inactive ADH). A similar phenomenon could be also observed in Acetobacter aceti, another genus of acetic acid bacteria. Furthermore, aeration conditions were also shown to affect ADH production; the ADH level was increased and was present as an active form under low-aeration conditions, while the ADH level was decreased and was present mainly as an inactive form under high-aeration conditions. Inactive ADH was solubilized from the membranes of G. suboxydans grown in acidic and high-aeration conditions and was purified separately from the normal, active form of ADH (active ADH). In spite of having 10 times less enzyme activity than active ADH, inactive ADH could not be distinguished from active ADH with respect to their subunit compositions, molecular sizes, and prosthetic groups. Inactive ADH, however, had a relatively loose conformation with a partially oxidized state, while active ADH had a tight conformation with a completely reduced state, suggesting that inactive ADH may lack a right subunit's interaction and that one of the heme c components may be inactivated. Reactivation from such an inactive ADH occurred either by shifting of the pH of the culture medium up during the cultivation or by incubation of the resting cells at the neutral pH region in the presence of an energy source such as D-sorbitol. Such an activation of ADH was repressed by the addition of a proton uncoupler and could not occur in the spheroplasts. Thus, the results suggest that inactive ADH could be generated abundantly under acidic growth conditions and converted to the active form at a neutral culture pH. The data also suggest that some periplasmic component may be involved in the conversion of inactive ADH into the active form by consuming some forms of energy.
醋酸菌的乙醇脱氢酶（ADH）是一种与膜结合的醌血红蛋白 - 细胞色素c复合物，参与醋的生产。在酸性生长条件下生长的氧化葡萄糖杆菌中，发现膜中ADH的含量大幅增加，但活性变化不大，这表明这种条件会产生无活性形式的ADH（无活性ADH）。在醋酸菌的另一个属——醋化醋杆菌中也观察到了类似现象。此外，通气条件也显示会影响ADH的产生；在低通气条件下，ADH水平升高且以活性形式存在，而在高通气条件下，ADH水平降低且主要以无活性形式存在。无活性ADH从在酸性和高通气条件下生长的氧化葡萄糖杆菌的膜中溶解出来，并与正常的活性形式的ADH（活性ADH）分开纯化。尽管无活性ADH的酶活性比活性ADH低10倍，但在亚基组成、分子大小和辅基方面，无活性ADH与活性ADH无法区分。然而，无活性ADH具有相对松散的构象和部分氧化状态，而活性ADH具有紧密的构象和完全还原状态，这表明无活性ADH可能缺乏正确的亚基相互作用，并且血红素c成分之一可能失活。这种无活性ADH的再激活可以通过在培养过程中将培养基的pH值上调，或者通过在存在能量源（如D - 山梨醇）的情况下将静止细胞在中性pH区域孵育来实现。添加质子解偶联剂会抑制ADH的这种激活，并且在原生质体中不会发生。因此，结果表明无活性ADH在酸性生长条件下可以大量产生，并在中性培养pH下转化为活性形式。数据还表明，一些周质成分可能通过消耗某种形式的能量参与无活性ADH向活性形式的转化。
